Lentivirus Packaging Protocol for Lentivirus Transfection
BioInnovatise Viral Vector Team
Updated December 1, 2024
What Is a Lentivirus Packaging Protocol and Which Is Right for My Research?
Transfecting cells with lentiviral vectors involves introducing the viral genome into the target cells, allowing for the expression of the transgene. A transfection protocol outlines the step-by-step procedure for introducing nucleic acids (such as plasmid DNA) into eukaryotic cells (such as a lentiviral vector). The goal of transfection is to temporarily or stably modify the cellular phenotype by delivering exogenous genetic material, i.e. payload. The type of protocol used can depend on the specific needs of the experiment, the characteristics of the cells, and the nature of the nucleic acids being introduced.
Lentivirus transfection protocols can vary for several reasons, depending on the specific experimental requirements, the characteristics of the lentiviral system being used, and the target cells for transduction. Important factors affecting the method of lentivirus transfection may include:
- Cell type
- Packaging cell line
- To learn more, read our article on lentivirus packaging cell line.
- Cell generation
- To learn more, read our article on lentivirus packaging generations.
- Packaging plasmids
- To learn more, read our article on lentiviral packaging plasmids.
- Purity requirements
- Expression systems
Our lentivirus packaging team will always use the specific transfection protocol requested by the customer. If you are unsure which lentivirus transfection protocol is right for you, contact our team. The below transfection protocol is a general protocol for educational purposes and should not be used outside of BioInnovatise productions.
Learn about our quick turnaround lentivirus packaging services.
The above diagram illustrates the lentivirus packaging process at BioInnovatise, where our team uses HEK293T cells for lentivirus packaging.
Materials Required
- Lentiviral vector stock
- Target cells
- Cell culture medium appropriate for the target cells
- Polybrene or other transfection enhancers (optional)
- Serum-free medium for transduction
- Incubator with appropriate conditions (temperature, CO², humidity)
Procedure
- Prepare cells
- Plate the target cells in a culture vessel the day before transfection to achieve 60–80% confluency on the day of transfection.
- Use appropriate cell culture medium for the specific cell type.
- Dilute lentivirus
- Dilute the lentiviral vector stock in serum-free medium. The optimal dilution will depend on the titer of the lentivirus and the desired multiplicity of infection (MOI). Typically, a range of MOIs should be tested to optimize transduction efficiency.
Transduction - Remove the culture medium from the cells. Add the diluted lentivirus to the cells. Optionally, you can add polybrene or another transfection enhancer at this step to improve transduction efficiency.
- Gently swirl or rock the culture vessel to ensure even distribution of the lentivirus.
- Dilute the lentiviral vector stock in serum-free medium. The optimal dilution will depend on the titer of the lentivirus and the desired multiplicity of infection (MOI). Typically, a range of MOIs should be tested to optimize transduction efficiency.
- Incubation
- Place the cells in the incubator and allow them to incubate for a suitable period. The incubation time can vary but is typically 6–24 hours. This allows the lentivirus to infect the target cells.
- Replace medium
- After the incubation period, replace the medium with fresh complete culture medium to remove any residual lentivirus.
- Monitor cells
- Incubate the cells for an additional period to allow for transgene expression. The time required will depend on the nature of the transgene and the experimental design.
- Harvest cells (if needed)
- Harvest cells for analysis or further experimentation. This may involve trypsinizing adherent cells or directly collecting suspension cells.
- Analytical techniques
- Analyze transgene expression using appropriate analytical techniques (e.g., fluorescence microscopy, flow cytometry, qPCR, Western blot) based on the nature of the transgene. Learn why GFP lentivirus is so prevalent in molecular biology for its inherent features.
Notes:
- Optimization: The transfection conditions may need optimization for different cell types and lentiviral vectors. This includes the MOI, incubation time, and the presence of transfection enhancers.
- Safety: Follow appropriate safety guidelines for working with lentiviral vectors, including the use of biosafety cabinets and personal protective equipment.
- Validation: Validate transduction efficiency and expression levels for your specific experimental goals.
- If you are using retrovirus vectors, our viral vector team can help determine which retrovirus packaging protocol is best for your research application.
Notes from the BioInnovatise laboratory:
- Our team uses HEK293T cells for lentivirus packaging
- Our team offers pseudotype coronavirus spike protein lentivirus, bald lentivirus lacking viral envelope protein, and integrase-deficient lentivirus cell options
- We use RT-qPCR (Real-time polymerase chain reaction) to measure titer levels
- We currently offer 2nd generation lentivirus and 3rd generation lentivirus packaging options
- Our preferred QC methods includes fluorescence microscopy, flow cytometry, and western blot.
Want to learn more about the latest in lentivirus based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News continuously collect and publish the latest information.
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